Structural and Functional Analysis of MtCIM Gene in Medicago truncatula

doi:10.13560/j.cnki.biotech.bull.1985.2023-0484

Abstract

Abstract:

【Objective】CIM（cytokinin induce messager）belongs to the Expansin protein family, which plays a critical role in plant growth and development. To analyze the structure, expression, subcellular localization, and function of MtCIM during Medicago truncatula growth and development. 【Method】Bioinformatics tools were employed to conduct a comprehensive study of molecular weight, theoretical isoelectric point, instability index, and protein structure prediction and analysis. Meanwhile, RT-qPCR method was used to measure the gene expressions under different hormone treatments（IAA, ABA and GA₃）. Fusion protein transient expression and yeast expression methods were used to identify its subcellular localization and transcriptional self-activation, respectively. Analysis of MtCIM-transgenic plants was performed in terms of their adaptation to a low phosphorus environment. 【Result】Bioinformatics analysis showed that the CIM protein had a relative molecular weight of 29.563 kD, theoretical isoelectric point of 5.54, and instability index of 41.62, indicating its protein property were unstable. The secondary and tertiary structure predictions showed that the protein was mainly composed of irregular curls. Exogenous IAA significantly increased the expression of MtCIM, while the expression of MtCIM showed a trend of rising and then decreasing after exogenous ABA and GA₃. The subcellular localization results showed that it was located in the cytoplasm. Upon analysis of transcriptional self-activation, it was found that there existed no self-activation activity in MtCIM. Analysis of the 20 obtained transgenic plants revealed that the cell walls of the leaves from these transgenic lines were thicker. Moreover, the transgenic plants demonstrated superior adaptability to low-phosphorus environments. 【Conclusion】A comprehensive analysis of the MtCIM gene in M. truncatula revealed its key role as a member of the Expansin protein family in plant growth and development. The phenotype of the transgenic plants demonstrated that MtCIM enhanced the adaptability of plants in low-phosphorus environments. These findings provide new insights for further understanding the role of the Expansin protein family in plant growth and development.

Key words: Medicago truncatula, MtCIM gene, self-activation testing, expression analysis, subcellular localization, low phosphorus response

XIE Hong, ZHOU Li-ying, LI Shu-wen, WANG Meng-di, AI Ye, CHAO Yue-hui. Structural and Functional Analysis of MtCIM Gene in Medicago truncatula[J]. Biotechnology Bulletin, 2024, 40(1): 262-269.

Figures/Tables 9

References 29

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引物名称 Prime name	引物序列 Prime sequence（5'-3'）
T7	TAATACGACTCACTATAGG
3-BD	TAAGAGTCACTTTAAAATTTGTAT
pGBKT7-CIM-F	GCTATTTCTACTGATTTTTCATGGCTCTTACACTTGAACA
pGBKT7-CIM-R	CAAAATCATGTCAAGGTCTTTTAAAAATTCACAATTGACC
3302Y-F	TGACGCACAATCCCACTATCCTT
3302Y-R	CCGTCCAGCTCGACCAGGAT
3302Y-MtCIM-F	GAACACGGGGGACTCTTGACCATGGCTCTTACACTTGAACA
3302Y-MtCIM-R	ACGCGTACTAGTCAGATCTACAAAATTCACAATTGACCTAT
MtActin-F	TGATCTGGCTGGTCGTGACCTTA
MtActin-R	ATGCCTGCTGCTTCCATTCCTAT
MtCIM-RT-F	TGGTGACGGAAGCGAAGGTGGTG
MtCIM-RT-R	CTTAACAGGGTTGCCTGAACATA

引物名称 Prime name	引物序列 Prime sequence（5'-3'）
T7	TAATACGACTCACTATAGG
3-BD	TAAGAGTCACTTTAAAATTTGTAT
pGBKT7-CIM-F	GCTATTTCTACTGATTTTTCATGGCTCTTACACTTGAACA
pGBKT7-CIM-R	CAAAATCATGTCAAGGTCTTTTAAAAATTCACAATTGACC
3302Y-F	TGACGCACAATCCCACTATCCTT
3302Y-R	CCGTCCAGCTCGACCAGGAT
3302Y-MtCIM-F	GAACACGGGGGACTCTTGACCATGGCTCTTACACTTGAACA
3302Y-MtCIM-R	ACGCGTACTAGTCAGATCTACAAAATTCACAATTGACCTAT
MtActin-F	TGATCTGGCTGGTCGTGACCTTA
MtActin-R	ATGCCTGCTGCTTCCATTCCTAT
MtCIM-RT-F	TGGTGACGGAAGCGAAGGTGGTG
MtCIM-RT-R	CTTAACAGGGTTGCCTGAACATA

顺式元件Cis-element	基序Motif（5'-3'）	位置Position	功能Function
ABRE	CACGTG	348	脱落酸响应元件ABA response element
	ACGTC	349
	ACGTC	708
P-box	CCTTTTG	53	赤霉素响应元件Gibberellin response element
TC-rich repeats	GTTTTCTTAC	103	防御和胁迫反应相关的元件Defense and stress response related elements
TGA-element	AACGAC	510	生长素响应元件Auxin response element
TCT-motif	TCTTAC	103	光响应元件Light response element
ARE	AAACCA	567	厌氧诱导元件Anaerobic induction element
TATA-box	TATTTAAA	21	转录起始点-30附近的核心启动子元件 Core promoter element near trabscription starting point -30
	TATAA	148
	TATTA	633
	TATA	502
	TATATA	87
	TATA	275
G-BOX	CACGTG	348	参与光反应的顺式作用元件Cis-acting elements involved in photoreactions
G-BOX	CACGTG	707

顺式元件Cis-element	基序Motif（5'-3'）	位置Position	功能Function
ABRE	CACGTG	348	脱落酸响应元件ABA response element
	ACGTC	349
	ACGTC	708
P-box	CCTTTTG	53	赤霉素响应元件Gibberellin response element
TC-rich repeats	GTTTTCTTAC	103	防御和胁迫反应相关的元件Defense and stress response related elements
TGA-element	AACGAC	510	生长素响应元件Auxin response element
TCT-motif	TCTTAC	103	光响应元件Light response element
ARE	AAACCA	567	厌氧诱导元件Anaerobic induction element
TATA-box	TATTTAAA	21	转录起始点-30附近的核心启动子元件 Core promoter element near trabscription starting point -30
	TATAA	148
	TATTA	633
	TATA	502
	TATATA	87
	TATA	275
G-BOX	CACGTG	348	参与光反应的顺式作用元件Cis-acting elements involved in photoreactions
G-BOX	CACGTG	707